We have developed a system to examine the consequences of a site-specific double-strand break (DSB) at a YZ junction in dispensable DNA within S. cerevisiae. A galactose-inducible HO-endonuclease cuts a YZ site placed in a plasmid (YZ-CEN) or at various positions within a YAC containing human DNA (YAC12). A persistent, long-lived DSB led to G-2 arrest and lethality. This indirect lethality was under genetic control since deletion of RAD9 resulted in higher viability. Most site-specific breaks in the YACs were rapidly repaired and did not lead to arrest or lethality, nor did a break in the YZ-CEN plasmid when it was rapidly degraded or repaired. By examining different strain backgrounds we have found differences in the genetic control(s) responsible for indirect lethality from a persistent DSB. A persistent DSB in a lambda DNA containing YAC (VS8) or the YAC12 derivatives, u8 or u17, did not induce cell cycle arrest or lethality in strain LS20. However, both cell cycle arrest and lethality resulted from a persistent DSB in strains NR85 (VS8) and CBY (u8 and u17). We, therefore, examined whether the presence of a high-copy yeast genomic library or a galactose-inducible human testis cDNA library could lead to indirect lethality by a persistent DSB in LS20. Seven yeast genomic fragments and two human cDNAs have been identified that enhance lethality from a persistent DSB. One yeast library clone (54-45) has been rescued into E.coli, sequenced and retransformed into LS20 containing either the u8 or u17 YACs. This clone contains 6 kb of DNA from chromosome III including the silent mating cassette HMR. In strain CBY, the YAC telomeric marker HIS3 is fully expressed; however, it is unexpressed (silenced) in LS20. Chromatin structure may therefore play a critical role in the expression of indirect lethality from a YAC DSB.